Exam 2 GoodNotes Biotransformation and Liver Toxicity

Oxidative N-Dealkylation

• Removes alkyl groups from N substituents like methyl, ethyl, n-propyl, isopropyl, n-butyl, allyl, and benzyl.
• Occurs on smaller alkyl groups first.
• 3° amines dealkylated to 2° amines faster than 2° amines to primary amines.
• Oxidation at the carbon $\alpha$ to the nitrogen.
• Similar to deamination.

N-Dealkylation

• Metabolite has been N-dealkylated.
• Requires H for demethylation to "nor".

Propranolol

• Has two $\alpha$ carbons.

Reactions

• Deamination and N-Dealkylation.

O-Dealkylation

• Similar mechanism to N-dealkylation.
• Hydroxylation on carbon attached to oxygen, followed by C-O cleavage.

Oxidative Deamination

• Compound A cannot undergo oxidative deamination, because it has "no H".

Codeine to Morphine

• O-Dealkylation converts codeine to morphine.
• CYP2D6 metabolizes 10% of codeine to morphine.
• Clinical consequences depend on CYP2D6 activity.
• Codeine metabolized to morphine can be fatal in kids & nursing mothers

Oxidation of Functional Groups

• Involves carbon-oxygen bonds.

Carbon-Sulfur Systems

1. Oxidative S-dealkylation (same mechanism as N and O-dealkylation)
2. Desulfuration
3. S-Oxidation - Catalyzed by FMO (and CYPs)
4. S-dimerization- catalyzed by FMO

Products

• Sulfoxide to sulfone.

Alcohol Metabolism

• Final product of alcohol metabolism is carboxylic acid.
• If a patient is deficient in ALDH, aldehyde will build up after consuming ethanol, it's not a good idea to drink alcohol.

Omeprazole S-oxidation

• Catalyzed by CYP3A4.
• Ritonavir inhibits CYP3A4, so when given with Drug X (also metabolized by CYP3A4), Drug X concentrations go up.

N-Oxidation

• Direct oxidation on Nitrogen.
• Secondary or tertiary amines by CYP450 or FMO.
• Primary amines by CYP450.
• Does not need an $\alpha$ H.

Reductive Reactions

• Warfarin and Nitrofurantoin reduction.

Enzymes

• Ketoreductase and Nitroreductase in Bacteria.

Azoreductase

• $RN=NR \rightarrow RNH<em>2 + RNH</em>2$

Hydrolysis

• Esters to carboxylic acids and alcohols, phenols.
• Amides to amines.
• Esterases and Amidases.

Aspirin Hydrolysis

• Aspirin to Salicyclic Acid and Acetic acid by esterase.
• Worst place to store aspirin is one where the smell is noticeable.

Amide Hydrolysis

• Nucleophile attacks at position C.

Valacyclovir

• Valacyclovir is converted to acyclovir via esterase or amidase.
• The red OH came from water.

Flecanide N-oxidation

• N-oxidation occurs by FMO and produces Metabolite A.

Conjugation Reactions

• Act on parent drug or Phase I metabolite.
• Purpose: links drug to polar endogenous compound for excretion.
• Increases water solubility and detoxifies.
• Involves enzymes and coenzymes.
• Different isoforms change or increase reaction.

Types

• Glucuronic acid, sulfate, amino acid, acetylation, methylation, glutathione conjugation.

Glucuronidation

• Most common Phase II reaction (lots of glucuronic acid in the liver).
• Direct condensation of drug with uridine diphosphate glucuronic acid (UDPGA).
• Reacts with -OH, -COOH, -$NH<em>2$, -$NR</em>2$, -SH groups.
• Enzyme: UDP Glucuronyl transferase.
• SN2 reaction with $\alpha$ or $\beta$ link.

Propylthiouracil Glucuronidation

• Step 1 is allylic or aromatic oxidation (or O-demethylation).
• Step 2 is glucuronidation.

Sulfate Conjugation

• Functional groups: alcohols, aromatic amines, N-hydroxy compounds.
• Both glucuronidation and sulfate conjugation can occur on the same substrates.
• Sulfate conjugation predominates in children up to age 9 (vs glucuronidation in adults).
• Co-enzyme: 3' phophoadenosine-5' phosphosulfate (PAPs).

Sulfate Conjugation

• Enzyme: sulfotransferases

Albuterol Sulfate Conjugation

• Three OH groups can undergo sulfate conjugation.

Amino Acid Conjugation

• Of carboxylic acids leads to amide bond formation.
• Amino acids: Glycine, glutamine, arginine, asparagine, histidine, lysine, glutamate, aspartate, alanine, and serine.
• Drug activated by ATP and coenzyme A (CoA) to form an acyl-CoA complex in mitochondria of liver and kidney cells.

Glutamine Conjugation

• Uses glutamine N-acyltransferase.

Nucleophile

• Amino group.

Glycine Conjugation

• Uses glycine N-acyltransferase.

N-Acetylation

• Primary amino group converted to uncharged amide (less soluble).
• Extent is genetically determined.
• Three phenotypes: homozygous fast, homozygous slow, heterozygous (intermediate) acetylators.
• Requires aliphatic or aromatic drug.
• Involves hydrazines/hydrazides.
• Enzyme: N-acetyltransferase.

N-dealkylation

• Products of N-dealkylation are B.

Glutamine Conjugation

• Aspirin cannot be directly metabolized through glutamine conjugation.

Phase II sulfate conjugation

• Is represented by path B.

Structure

• Is a co-enzyme for sulfate conjugation.

Amino acids

• Linked to form a peptide through amides.

Sulfate Conjugation

• The drug can directly undergo sulfate conjugation (without any Phase I metabolism first).

N-Acetylation Mechanism

• Coenzyme Acetyl CoA

Methylation

• Biosynthesis of endogenous compounds.
• Minor metabolic pathway.
• Enzyme: Methyltransferase.

Types

• O-Methylation: Catalyzed by Methyltransferases; e.g., Catechol-O-methyltransferase (COMT) for catechol metabolism.
• N-Methylation: Methylation of primary and secondary amines.
• S-Methylation: Detoxification step for thiols.

Methylation

• S-Adenosylmethionine (SAM) is the coenzyme.

Catechol O-Methylation

• COMT performs O-methylation.
• N-Methyltransferase performs N-methylation.

Structure of Norepinephrine

• Structure A is norepinephrine.

Glutathione Conjugation

• Electrophilic drugs/xenobiotics react with GSH to form S-substituted GSH adducts.
• GSH tripeptide (g-glutamyl-cysteinyl- glycine).
• Undergoes further transformation to N-acetylcysteine products called mercapturic acids.
• A detoxifying pathway.

GSH

• Is a tripeptide chain.

GSH and Mercapturic acid conjugates

1. Initial conjugation
2. Removal of $\gamma$-Glu and Gly
3. N-acetylation

Glutathione

• Very water-soluble and highly reactive.

Acetaminophen

• Can form mercapturic acid metabolite.

What Happens If We Deplete GSH?

• S-Liver protein is formed.

Ethacrynic Acid

• Glutathione adduct of Ethacrynic Acid can be formed.

Cysteine

• Michael acceptor.

Treatment for Overdose

• Administer acetylcysteine.

O-methylation

• Requires S-adenosylmethionine as a co-enzyme.

Glucuronidation

• Requires UDP Glucuronyl transferase as an enzyme.

Biotransformation and Liver Toxicity Learning Objectives

• Review relevant liver anatomy and physiology.
• Review biotransformation of drugs.
• Discuss drug-induced liver injury.
• Describe the mechanisms involved in drug-induced liver injury using Valproic acid and Tylenol.

Liver

• Important organ for metabolism

Biotransformation

• Metabolic processes that facilitate excretion of substances from the body.
• Involves Xenobiotics (natural & synthetic, lipophilic).
• Phase I: oxidation, reduction, hydrolysis (reactive, hydrophilic).
• Phase II: conjugation (detoxified, hydrophilic).
• “Phase III”: specialized transporters (OATP, BSEP, MRP).
• Located at portals of entry and exit (Alimentary Tract, LIVER, Lungs, Kidneys, Upper Respiratory Tract, Skin).

The Liver

• Susceptible to Toxicity due to: biotransformation enzymes, uptake of nutrients/xenobiotics, biliary secretion, anatomic location.
• Central metabolic organ.
• Hepatocytes transform dietary nutrients into fuels and precursors required by other tissues.
• Also transforms xenobiotics into toxic/non-toxic metabolites for elimination.
• Can adjust enzyme levels to adjust to nutritional state.
• Detoxifies foreign organic compounds (Cytochrome P-450 and others).
• regenerates itself.

Liver Circulation

• Hepatic vein to inferior vena cava is the entry to systemic circulation.
• The Liver receives bile, hormones, nutrients, pathogens, bacteria, viruses, and toxins.

Enterohepatic Cycling

• Orally administered Rx undergoes GI absorption.
• The Liver performs glucuronide formation (phase II metabolism).
• Conjugates (>500Da) in bile pass to intestine.
• Intestinal hydrolysis of conjugates (beta-glucuronidase) occurs.
• Hydrolyzed drug gets re-absorbed, resulting in elevated or prolonged blood levels.

Hepatic Lobule

• The portal triad contains a hepatic artery (O2 to liver), portal vein (systemic blood to liver), and bile duct (bile to gallbladder).
• Blood arrives to the lobule via the portal vein and the hepatic artery.
• Blood is dumped into the sinusoids (capillaries with large “fenestrae”).
• Hepatocytes (and other cells) surrounding the sinusoids have access to the blood and can “clean it up!”.
• CV = Central Vein (“cleaned up blood back to systemic circulation).
• Blood and Bile flow Opposite.
• Kupffer cells are present.

Biotransformation Location In Liver Lobule

• Periportal: enriched with glutathione, oxygen, intermediary metabolism enzymes, and alcohol dehydrogenase.
• Centrilobular is enriched with Mixed-Function Oxidases (MFOs) → CYPs.

Biotransformation Location Liver Cells

• Hepatic parenchymal cells (Hepatocytes) participate in Biotransformation.
• Smooth Endoplasmic Reticulum, Mitochondria (microsomes), Cytosol.
• Disrupt Biochemical/Physiological Function → negative consequences.

Liver Injury

• Drug-Induced Liver Injury (DILI).
• Necrosis: cell swelling, nuclear disintegration, inflammation.
• Apoptosis: cell shrinkage, nuclear fragmentation, programmed cell death, without inflammation.
• Assay for leakage of alanine aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) or gamma-glutamyltransferase (GGT).
• Jaundice (most common sign).
• Canalicular cholestasis (decreased bile flow from the liver to intestine).
• Bile duct damage.
• Steatosis (fatty liver – disrupted metabolism - NASH).
• Fibrosis/cirrhosis - collagen scars from Hepatic Stellate Cells.
• Tumors.
• Hepatitis – inflammation of the liver – Kupffer cells (macrophages).
• Liver rapidly regenerates tissue and function – Discontinue the medication!

Drug-Induced Liver Injury (DILI)

• ~50% of cases of acute liver failure are caused by drugs.
• ~750 marketed drugs have risks of causing DILI.

1. Intrinsic DILI: dose-dependent, predictable and reproducible; quick
2. Idiosyncratic DILI: Determined by the interaction of host, drug and environment factors; (unique to an individual); longer latency

Clinical Manifestations/Diagnostic Criteria of DILI

• Can mimic acute and chronic liver diseases!
• Symptoms: fever, nausea, vomiting, jaundice, dark urine, itching and right upper quadrant pain.
• Certain Drugs have “signature injury patterns”.

Clinical Manifestations/Diagnostic Criteria of DILI

• Hy’s Law:

1. ALT, AST 3X ULN
2. Serum total bilirubin 2X ULN with NO cholestasis (ALP < 2X ULN)
3. Drug ONLY explanation of test results!

Factors Affecting Drug Metabolism/Liver Toxicity

• Influenced by estrogen, alcohol, obesity, smoking, dose, induction, inhibition, Vitamin/Mineral Deficiencies, SNPs, Racial/Ethnic Diff., Nearness to toxic environments.

Mechanisms of Liver Toxicity

• Toxic Chemicals/Metabolites Disrupt Biochemical/Physiological Function → negative consequences.

Mitochondria-Induced Apoptosis

• Biotransformation happens in the cytoplasm, mitochondria, and the endoplasmic reticulum – which means stress and toxicity can also happen here.
• Apoptosis: programmed cell death – highly regulated and controlled. Sometimes good, sometimes bad.
• Intrinsic pathway: cell kills itself because it senses cell stress.
• Extrinsic pathway: signals from somewhere else (other cells, etc).
• Some signals for Apoptosis: increased intracellular and mitochondrial concentration of free fatty acids, increased intracellular and mitochondrial calcium, and oxidative stress.
• Drugs can interfere with proper metabolic processes, which causes dysfunction of metabolic processes that hurts the mitochondria and triggers apoptosis.

ROS, Ca2+, FFA

• Reactive Oxygenated Species (ROS), Free Fatty Acids (FFA), and Cytochrome c are important.
• The Drug, or its metabolite, disrupts normal biochemical pathways in liver mitochondria.

Mechanism of valproic acid-induced liver injury

• Is mitochondria dysfunction from increased FFA and ROS.

Impaired Lipid Metabolism: Valproic Acid

• Used to treat bipolar disorder, epilepsy, and as a preventative for migraines.
• Valproic Acid (Valproate) is a branched, short-chain fatty acid that can freely enter the mitochondria.

Metabolized:

• Conjugation (UGT enzymes: non-toxic metabolites)
• Beta-Oxidation
• Omega-Oxidation
• Some CYP metabolism

Actions

1. VPA enters mitochondria, gets –CoA’ed, Blocks Carnitine Shuttle, Beta-Oxidation, Depletes CoA Stores, Fatty Acids cannot enter mitochondria, increase FFA concentration in cells, Fatty Acids in the mitochondria do not get metabolized, increase FFA concentration in mitochondria.
2. VPA gets CYP metabolized in cytosol, reactive metabolite enters mitochondria, blocks Beta-oxidation enzymes, and therefore Beta-Oxidation itself.
3. Omega-Oxidation of VPA → toxic metabolites that block the Urea Cycle and lead to hyperammonemia.

Lipid Metabolism leading to liver toxicity?

• Accumulation of Free Fatty Acids in the mitochondria and in the cytoplasm of hepatocytes (fatty liver (steatosis)).
• Accumulation of FFAs in the mitochondria of hepatocytes leads to Decreased CoA, Inhibition of the Electron Transport Chain and Citric Acid Cycle, Decreased ATP, decreased production of molecules necessary for anabolism Increased ROS, Apoptosis.
• Energy Deficiency in Extra-Hepatic Tissues.
• Apoptosis can also signal Hepatic Stellate Cells to synthesize collagen scars → Fibrosis/Cirrhosis.

Valproic Acid Toxicity: Summary

• High Likelihood.
• Enzyme elevations, severe liver injury with progressive jaundice, coma and death!
• Monitor.
• Treatment: Remove the drug, Dialysis, Liver Transplant, Antidote: Carnitine (why?).
• Satisfies Hy’s Law. Valproic Acid and its metabolites inhibit Fatty Acid Oxidation, leading to a disruption of mitochondrial function, apoptosis, steatosis, fibrosis/cirrhosis, and hepatitis.

Reactive Oxygen Species (ROS)

• Free Radicals, and Oxidative Stress.
• Free Radical: Unpaired electron in outer shell. Properties differ from parent compound among radicals.

Sources

• Endogenous – Inflammation and reactive CYP metabolites, biochemical pathways.

• Exogenous – ionizing radiation, halogenated compounds, and metal ions (catalysts).

• NOT ALL ROS IS BAD. For example, physiological concentrations of ROS are necessary for synaptic plasticity and normal cognitive function. ROS can initiate inflammatory processes.

ROS in Danger for Cell

• Radical reactions deplete cellular energy and anti-oxidation systems.
• Acceleration of Reaction: free radical generation exacerbates free radical generation.

Active Species

• Superoxide radical, hydoxy and hydroperoxy radicals target cells or macrophages.

Oxidative Injury

• Re-dox cycling makes O radicals and uses anti-oxidants.
• Free Radical Attack.
• Lipid Peroxidation.
• Oxidative Stress to Protein (enzyme) targets (homeostasis).
• Acetaminophen Toxicity!

Free Radical Pathology

• •$O_2^- + HOOH$
• •$O<em>2^- + 2H^+ \rightarrow O</em>2$ (Superoxide Dismutase)
• •$OH^-$
• Fenton Reaction Fe2+, Cu1+, Cr5+, Ni2+, Mn2+.
• Catalase (in peroxisomes)
• Glutathione peroxidase.
• 2GSH GSSG 2HOH

Glutathione Peroxidase

• cytosol (selenoenzyme).
• Oxidized, must be reduced again!
• Reduced form (GSH) is the antioxidant!

Acetaminophen

• Is linked to Oxidative Stress. Acetaminophen (Tylenol).
• > 25 billion doses sold annually in the US.
• Most common cause of Acute Liver Failure (ALF) → ~50% of cases.
• Doses exceeding 4g/day.
• Intentional: suicide.
• Unintentional: many medications contain acetaminophen. Patients don’t pay attention → “therapeutic misadventures”.
• Predisposition for toxicity: increased P450 metabolism, depleted glutathione.

DILI

• Glutathione (-), Alcohol depletes/induces,NAPQI is a strong oxidizer!, it must be reduced,causes oxidative stress and Glutathione reduces it!

GHS and Mercapturic acid

• Glutathione is first conjugated, then $\gamma$-Glu and Gly are removed for the ultimate N-acetylation process.

Oxidative Injury

• From NAPQI. What is NAPQI doing? Depleting Glutathione.

Glutathione Depletion

• GSH is the reduced form (antioxidant).
• Glutathione transferases (Phase II).
• Formation of glutathione conjugates.
• Oxidative injury to cell.
• GSH oxidized to GSSG (glutathione disulfide).
• Must be reduced again to work!
• NADPH from the Pentose Phosphate Pathway.

Acetaminophen: So What?

• It's necessary to regulate.
• Acetaminophen is metabolized into a toxic compound (NAPQI) that causes oxidative stress and can’t be cleaned up if Glutathione is depleted.

Oxidative Stress Interventions

• ANTIOXIDANT ACTIVITY.
• Vitamin C, Vitamin E, Deferroxamine (metal chelator), Herbals.
• Glutathione mono or diesters,carotenoids, resveratrol (red wine), green tea, etc.

How Does Glutathione Stop NAPQI?

• Increased ROS caused by Angelman Syndrome is eliminated when treated with MitoQ.

Oxidative Stress Summary

• ROS (generated) causes Oxidative Damage to DNA,Protein and Lipid.

Pharmacology

• Study of substances (drugs) that has an ability to affect a living organism.
• Pharmacodynamics effects.
• Pharmacokinetics ADME parameters.

Classification of Drugs

• Agonists vs Antagonists, they can be Direct-Acting or Indirect-Acting. Agonists have AFFINITY and EFFICACY while Antagonists only have AFFINITY.

Quantitative Aspects

• Occupy receptors and Produce a desired/toxic response
• Helps us to compare potency and efficacy of drugs
• Helps us to determine the dose range for patients
• Drug/dose/concentration binding curves (DBC/CBC)
• Dose/concentration response curves (DRC/CRC)

Drug/Dose Binding Curves

• basis: increasing concentration of drug increases the number of receptors occupied by the drug
• $Kd$ = concentration of the drug required to bind 50% of the receptors

DBC/CBC

• Kd: affinity of the drug, low Kd = high affinity
• Bmax: total number of receptors occupied by drug
• doesn´t tell us the nature of the drug and of the biological response.
• Drug development and basic research.

Dose Response Curves

• Basis: increasing concentration of drug increases the magnitude of effect.
• Graded DRCs: response of an individual.
• Quantal DRCs: response of a population.

Graded Dose Response Curves

• Efficacy (Emax): maximal response produced by the drug.
• Potency (EC50): concentration that produces 50% of the maximal response.
• Threshold concentration (Tc): concentration below which no effect is observed.
• Slope (h).

Types of Agonists

• Direct-acting agonists: act on receptor.
• Indirect-acting agonists: ↑ NT levels.
• Direct-acting agonists can act on the active or the allosteric site of the receptor and can be:
  • Full agonists
  • Partial agonists
  • Inverse agonists
  • Allosteric agonists

Allosteric agonists

• Decrease $EC_{50}$,potency increases..

antagonists

• can be noncompetitive or competitive.

Quantifying Toxicity: DRCs

• Toxicity $\propto$ dose

Express Dose

• Single/MUltiple mg kg, mg/M2, mg/mouse, mg/M2/day.

Quantal toxicity

• ${LD}_{50}$=lethal dose 50%.
• NED = Normal Equivalent Deviate.

Understanding Safety

• Therapeutic Index TI
• Margin of safety MoS

Variation in Toxic Responses

• Selective toxicity.
• Species differences.
• Individual differences.